Zebrafish Embryonic Manipulation and Analysis

DMH1 (Sigma-Aldrich, St. Louis, MI, United States, D8946) used in experiments was reconstituted at stock concentrations (10 mM) in DMSO solvent. Embryos were dechorionated by hand using forceps and dechorionated embryos were transferred to 6-well plates. Embryo media with appropriate drug concentration or vehicle control was added to each well. For DMH1 treatments, embryos were treated from 28 hpf to 76 hpf. Embryos were incubated at 28.5°C for the duration of the drug treatment. For phenotypic analysis, embryos were mounted in 2% methylcellulose and documented using Leica M165FC microscope and Leica MC 170 HD camera. P values were calculated using unpaired t-test with Welch’s correction (GraphPad Prism7).
The sequence of alk3a, alk3b, alk6a, and alk6b morpholinos were used as previously described (Little and Mullins, 2009 (link); Neumann et al., 2011 (link)) (Supplementary Table 1). Each morpholino was injected into 1-cell stage wild-type zebrafish embryos. The morpholino doses used are 1, 2, 4, 8, 16, and 32 ng. Embryos were collected at 52 and 76 hpf and mounted in 2% methylcellulose and documented using Leica M165FC microscope and Leica MC 170 HD camera.